1. Extraction of Human DNA from blood

2. Experiment Goals Isolation of genomic DNA from human blood
Analysis of isolated DNA using
Agarose gel electrophoresis
Spectrophotometry or Nnophotometer

3. DNA Extraction
DNA extraction is the first step for subsequent molecular or forensic analysis.
DNA can be extracted from almost any intact cellular tissue
Skin,
blood,
saliva,
semen,
mucus,
muscle tissue,
bone marrow, etc.

4. Basic steps in DNA extraction
There are three basic steps in a DNA extraction, the details of which may vary depending on the type of sample and any substances that may interfere with the extraction and subsequent analysis.
Harvest the nucleated cells
Lysis of the cells and nuclei and digest/denature the proteins
Separate contaminants from DNA
Precipitate DNA .
Resuspend DNA in final buffer

5. Lysis step
Most mammalian cells and cultured cells can be lysed easily using detergents.
Organisms that have cell walls in addition to a cell membrane usually require treatment with additional enzymes to effectively disrupt the cell wall.
For example, lysozyme is used to digest the thick peptidoglycan cell wall of Gram positive bacteria

6. Lyticase (or zymolase) enzyme is used to disrupt the polysaccharide cell wall of yeasts.
For some plant species, mechanical disruption of tissues in addition to detergent treatment is sufficient.
For other plant species, use of cellulase enzyme is required to disrupt the cell wall.

7. DNA isolation DNA must be separated from proteins and cellular debris.
Separation Methods:
Organic extraction
Salting out
Selective DNA binding to a solid support

8. DNA Isolation Methods Liquid Phase Organic Extraction
Phenol :chloroform/isoamyl alcohol
This organic solvent is usually added to the sample and then, using centrifugal force, two layers are formed.
hydrophobic layer on the bottom, with the hydrophilic layer on top.
Lipids and other hydrophobic components will dissolve in the lower hydrophobic phase. DNA will dissolve in the upper aqueous phase.(DNA is polar and therefore insoluble in organic solvents)

9. Amphiphilic components, protein, which have both hydrophobic and hydrophilic properties at the interface between the two layers.
DNA is removed from top aqueous layer.
DNA is precipitated with alcohol or isopropanol and rehydrated.
Disadvantages:
Slow, labor-intensive, toxic (phenol, chloroform)
Fume hood required, disposal of hazardous materials required

10. DNA Isolation Methods: Liquid Phase Nonorganic Salt Precipitation
Inorganic DNA extraction is sometimes called “salting
out” It makes use of high salt conditions to selectively precipitate proteins, leaving the DNA in solution.
Proteins are precipitated with salt solution.
DNA is precipitated with alcohol or isopropanol and rehydrated.
Advantages:
Fast and easy method
Uses nontoxic materials, no fume hood required, no hazardous materials disposal issues
Produces high-quality DNA

12. DNA Isolation Methods Solid Phase Procedures
Uses solid support columns
Solid support columns: Fibrous or silica matrices bind DNA allowing separation from other contaminants.
Silica matrices have unique properties for DNA binding.

13. After the immobilized DNA is washed with buffer, the DNA is eluted in a specific volume of water, TE, or other low salt buffer.
Advantages:
Fast and easy, no precipitation required

14. Procedure

15. Overview of Procedure 1- Lyse RBCs and harvest the WBCS
2- Lyse WBCs nuclei & Denature/digest proteins
3- Separate contaminants (e.g., proteins, heme)
4- Precipitate DNA
5- Resuspend DNA in final buffer

16. Blood Collection Blood collected in disodium EDTA tube
to prevent in vitro blood clot formation.
Why prefer to use EDTA tube not heparin?
EDTA tubes provide good yields of nucleic acid appropriate for PCR and
other assays.

17. Heparin, on the other hand, is a problem when mixed with samples intended for nucleic acid extraction.
Heparin adsorbs to nucleic acid and is not completely removed by standard extraction techniques.
Residual heparin in a DNA or RNA sample can inhibit restriction digests, PCR, and other enzyme-based molecular biology assays.

18. The method for purification in this experiment
Liquid phase :Nonorganic Salt Precipitation

19. 1- RBCs Lysis
to isolate nucleated cells takes advantage of the differences in the osmotic fragility of RBCs and WBCs.
Incubation of whole blood in hypotonic buffer (cell lysis solution) or water will result in the lysis of the RBCs before the WBCs. The WBCs are then pelleted by centrifugation.
The result of this step:
White pellet is observed at bottom of tube
Repeat this step if the pellet don’t become white.

20. 2- WBCs nuclei Lysis & proteins digestion
Using Nucleic lysis solution:
SDS which disrupts the lipid layers, helps to dissolve membranes & denaturation of proteins. in some cases may Include a protease (proteinase K) to digest the proteins
EDTA which chelates Mg++ and is a co-factor of DNase which destroy up DNA rapidly,

21. 2- WBCs nuclei Lysis & proteins digestion
Using Nucleic lysis solution:
SDS which disrupts the lipid layers, helps to dissolve membranes & denaturation of proteins. in some cases may Include a protease (proteinase K) to digest the proteins
EDTA which chelates Mg++ and is a co-factor of DNase which destroy up DNA rapidly,

22. 3- Separate contaminants from DNA
By salting out method (protein precipitation solution)
4- Precipitate DNA
By isopropanol or 100%ethanol.
DNA will be precipitated DNA will be precipitated by gentle swirling & observed as a white thread like strand

23. The addition of ethanol 100% OR isopropanol to the aqueous phase containing DNA will result in the precipitation of the DNA, which can thus be subsequently sedimented by centrifugation.
The precipitate containing the DNA is washed with 70 % ethanol in order to remove the salt content of the preparation

24. Ethanol Precipitation of DNA
DNA is polar, soluble in water (high polar) & Insoluble in less polar ethanol
1. 100 % ethanol
Centrifugation
Precipitate DNA & salts that form ionic bonds with DNA
no water molecules are free to dissolve DNA
2. 70% ethanol
30 % water solubilizes the salts present in the pellet
!!! Supernatant is removed
DNA is resuspended in TE / dH2O

25. 5- Resuspend DNA in final buffer
Dried pellet is resuspended in TE buffer and left overnight to dissolve the DNA pellet completely.

27. -Incubation for 10 minutes. Vortex many time through incubation period
-centrifugation rpm for 3min
-Mix by inversion (not vortex) after nuclei lysis solution
-vortex 20 sec after protein precipitation solution
centrifugation rpm for 3min
Mix by inversion (not vortex) after isopropanol .
Add ethanol (70%) as the same of isopropanol

28. Rehydrate the DNA in the appropriate volume of DNA Rehydration Solution for 1 hour at 65°C or overnight at 4°C.